Project Summary Transposable elements (TE) comprise roughly 40% of murine and human genomes. Loss of cellular silencing mechanisms leads to active transposition and human diseases like hemophilia and leukemia. Endogenous retroviruses (ERV) are one class of TE formed by the remnants of past retroviral infection that have accumulated in the genome over millennia. Transcription of ERV is upregulated in a variety of human and murine autoimmune and oncologic diseases, yet the mechanism and biological relevance of this association remains unclear. We have identified that expression of one class of ERV is markedly enhanced in CD4+ T lymphocytes of C57BL/6N mice relative to those of C57BL/6J mice, and determined that this phenotype is recessive. There are many phenotypic differences between C57BL/6N and C57BL/6J substrains; however, the genetic causes of these differences are largely unknown. Prior studies have extensively characterized coding- sequence variation between these substrains but the specific mechanisms that contribute to silencing of ERV transcripts in murine lymphocytes have not been characterized. We therefore aim to perform a classical genetic segregation study, followed by in vitro validation and functional characterization of the identified variant, in order to determine the genetic cause of this phenotype and to better understand the mechanisms governing endogenous retroviral silencing in circulating lymphocytes. In Aim 1, we will perform quantitative trait locus analysis of segregating C57BL/6N and C57BL/6J crosses to identify the coding- variant driving enhanced ERV expression in the C57BL/6N background. We will validate this variant in vitro using CRISPR/Cas9-mediated gene editing of the targeted nucleotide. In Aim 2, we will characterize chromatin accessibility, histone modification, and DNA methylation differences between C57BL/6N and C57BL/6J CD4+ T cells by performing assay for transposase-accessible chromatin (ATAC)-sequencing, chromatin immunoprecipitation-sequencing, and bisulfite-sequencing. In Aim 3, we will characterize the biochemical and functional effects of the identified SNP on its associated protein. We will correlate these epigenetic landscape differences to the functional changes mediated by the SNP of interest and thereby uncover a novel pathway of non-ecotropic ERV expression control.